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J. Biol. Chem., Vol. 275, Issue 51, 39811-39814, December 22, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/51/39811    most recent C000709200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, G. Articles by Clore, G. M. Search for Related Content PubMed PubMed Citation Articles by Wang, G. Articles by Clore, G. M.

ACCELERATED PUBLICATION
A Novel Membrane Anchor Function for the N-terminal Amphipathic Sequence of the Signal-transducing Protein IIA^Glucose of the Escherichia coli Phosphotransferase System^*

Guangshun Wang, Alan Peterkofsky^§, and G. Marius Clore^¶

From the  Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0510 and the ^§ Laboratory of Biochemical Genetics, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-4036

Received for publication, October 9, 2000

	ABSTRACT

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Enzyme IIA^Glucose (IIA^Glc) is a signal-transducing protein in the phosphotransferase system of Escherichia coli. Structural studies of free IIA^Glc and the HPr-IIA^Glc complex have shown that IIA^Glc comprises a globular -sheet sandwich core (residues 19-168) and a disordered N-terminal tail (residues 1-18). Although the presence of the N-terminal tail is not required for IIA^Glc to accept a phosphorus from the histidine phosphocarrier protein HPr, its presence is essential for effective phosphotransfer from IIA^Glc to the membrane-bound IIBC^Glc. The sequence of the N-terminal tail suggests that it has the potential to form an amphipathic helix. Using CD, we demonstrate that a peptide, corresponding to the N-terminal 18 residues of IIA^Glc, adopts a helical conformation in the presence of either the anionic lipid phosphatidylglycerol or a mixture of anionic E. coli lipids phosphatidylglycerol (25%) and phosphatidylethanolamine (75%). The peptide, however, is in a random coil state in the presence of the zwitterionic lipid phosphatidylcholine, indicating that electrostatic interactions play a role in the binding of the lipid to the peptide. In addition, we show that intact IIA^Glc also interacts with anionic lipids, resulting in an increase in helicity, which can be directly attributed to the N-terminal segment. From these data we propose that IIA^Glc comprises two functional domains: a folded domain containing the active site and capable of weakly interacting with the peripheral IIB domain of the membrane protein IIBC^Glc; and the N-terminal tail, which interacts with the negatively charged E. coli membrane, thereby stabilizing the complex of IIA^Glc with IIBC^Glc. This stabilization is essential for the final step of the phosphoryl transfer cascade in the glucose transport pathway.

	INTRODUCTION

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The signal-transducing protein IIA^Glucose (IIA^Glc)¹ is an integral component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Escherichia coli. Glucose transport via the PTS initiates from phosphoenolpyruvate, which auto-phosphorylates enzyme I. Enzyme I transfers the phosphoryl group to the histidine-containing phosphocarrier protein, HPr, which in turn donates a phosphoryl group to IIA^Glc. Subsequently, IIA^Glc transfers the phosphoryl group to the solvent-exposed IIB domain of the membrane protein (1). In addition to its role in the PTS, IIA^Glc also modulates the activity of a number of other proteins, depending on its phosphorylation state. Although dephosphorylated IIA^Glc is a negative regulator of glycerol kinase (2) and various non-PTS permeases (1), phosphorylated IIA^Glc is a positive regulator of adenylyl cyclase activity (3). Structural studies on phospho- and dephospho-IIA^Glc by both x-ray crystallography (4, 5) and NMR spectroscopy (6, 7) have shown that the protein is composed of a globular core (residues 19-168) comprising a predominantly -sheet sandwich and an unstructured N terminus (residues 1-18), which is invisible in electron density maps and highly mobile in solution. Recently, we determined the solution structure of the intact IIA^Glc-HPr complex by multidimensional NMR (8), and the N-terminal 18 residues remain disordered. Further, chemical shift mapping has shown that the N-terminal 18 residues are unperturbed upon binding of IIA^Glc to the isolated IIB domain of IIBC^Glc (9), and our current structural studies on the IIA^Glc-IIB complex indicate that the N-terminal segment of IIA^Glc remains unstructured and is not involved in this protein complex.² Biochemical studies, on the other hand, have shown that whereas the presence of an intact N-terminal segment of IIA^Glc is not necessary for the transfer of phosphorus from HPr to IIA^Glc it is absolutely required for effective phosphoryl donation from IIA^Glc to IIBC^Glc (10). The structural and functional role of the N-terminal portion of IIA^Glc, however, has not been elucidated. In this communication, we show that intact IIA^Glc and a synthetic peptide corresponding to the first 18 N-terminal residues of IIA^Glc interact with anionic phospholipids found in the membrane of E. coli, promoting the formation of an amphipathic helix. Based on these data, we propose a two-state model for IIA^Glc and discuss the significance of our observations in the context of sugar transport and the PTS in E. coli.

	MATERIALS AND METHODS

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Enzyme IIA^Glc from E. coli was expressed and purified as described (11) and quantified by UV spectroscopy at 257 nm. The N-terminal 18-residue peptide of IIA^Glc was synthesized by solid phase methods with the C-terminal end amidated and purified by reverse phase high pressure liquid chromatography (Commonwealth Biotechnologies, Inc.). The peptide was greater than 99% pure as judged by mass spectrometry and amino acid compositional analysis. Dioleoylphosphatidylglycerol (PG), phosphatidylethanolamine (PE) purified from E. coli membranes, and dioleolylphosphatidylcholine (dioleolyl-PC) were purchased from Sigma and were  98% pure. Vesicles were made by sonication of lipids in 10 mM phosphate buffer, pH 7, as reported elsewhere (12).

CD spectra were collected at 22 °C on a Jasco J-720 spectropolarimeter (calibrated using 0.06% d-(+)-10-camphorsulfonic acid at 290.5 nm) from 190 to 250 nm using a 2-mm path length cell, with a scan rate of 2 nm/min, a time constant of 0.5 s, a bandwidth of 1 nm, and a sensitivity of 20 millidegrees. Each spectrum is the average of 10 scans. After smoothing and background subtraction, the spectrum was expressed in molar ellipticity.

	RESULTS AND DISCUSSION

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The amino acid sequence of the N-terminal 18 residues of E. coli IIA^Glc is Met-Gly-Leu-Phe-Asp-Lys-Leu-Lys-Ser-Leu-Val-Ser-Asp-Asp-Lys-Lys-Asp-Thr. According to the convention adopted for E. coli IIA^Glc in the literature (1, 4-8), the N-terminal methionine, which can be hydrolyzed, is numbered as zero. An interesting feature of this sequence is the periodical occurrence of hydrophobic (italicized) and hydrophilic amino acid residues. Such periodicity, previously reported in human apolipoproteins (13, 14), is correlated with the potential to form an amphipathic helix. When the sequence is represented on a helical wheel projection (Fig. 1), it is apparent that there is a cluster of hydrophobic residues on one side of the wheel (Leu², Phe³, Leu⁶, Leu⁹, and Val¹⁰) and a cluster of hydrophilic residues on the other (Asp⁴, Ser⁸, Ser¹¹, and Asp¹²). Located at the boundary of the hydrophobic and hydrophilic faces are two lysine residues (Lys⁵ and Lys⁷). Such features are reminiscent of the class A amphipathic helix in apolipoproteins (14). Because apolipoproteins bind lipids, we reasoned that the N-terminal sequence of IIA^Glc may interact with lipids in the E. coli membrane.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Helical wheel projection of the sequence corresponding to the N-terminal 18 residues of E. coli IIAGlc. In accordance with the nomenclature in the literature (1, 4-8), the N-terminal Met of IIAGlc is numbered as zero. Hydrophobic residues are colored in blue, interfacial cationic residues in red, and hydrophilic residues in black. Residues Asp13-Lys14-Lys15-Asp16-Thr17 in parentheses, which do not fit the helical wheel projection, are proposed to be in the linker region between the two domains of IIAGlc when associated with the membrane (see Fig. 3).

To test this hypothesis, we investigated the interaction of a synthetic peptide comprising the N-terminal 18 amino acid residues of IIA^Glc (referred to as Pep18) with a variety of phospholipid vesicles. Fig. 2a presents the CD spectra of Pep18 in the presence or absence of anionic lipids. In the absence of lipid (green), there is a strong negative band at 198-200 nm, indicative of a random coil (15), consistent with its disordered structure in intact IIA^Glc (4-8). In the presence of the anionic lipid PG (red), however, there is a dramatic change in the CD spectrum of Pep18, which displays double minima at ~208 and ~222 nm, characteristic of an -helix (12, 16). The helicity was estimated to be ~50% based on 222-nm band analysis (17). In the context of the helical wheel projection shown in Fig. 1, this suggests that the helical segment comprises residues 2-10. In contrast, essentially no change was seen in the CD spectrum of Pep18 upon addition of the zwitterionic lipid dioleolyl-PC under the same conditions (data not shown). The observation that Pep18 interacts with an anionic but not zwitterionic lipid indicates that electrostatic interactions between the anionic lipid head groups and cationic lysine side chains of Pep18 play a significant role in -helix stabilization (12, 18, 19). In this regard, it is noteworthy that the predominant lipids in the membrane of E. coli are anionic, comprising ~25% PG and ~75% PE (20). Indeed, Pep18 also forms an -helix in a 1:3 PG:PE mixture (Fig. 2a, black) with near identical helicity to that observed with PG alone.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   CD spectra of (a) 52 µM Pep18 and (b) 13 µM IIAGlc in the presence and absence of 2 mM PG, pH 7, and 22 °C are shown. The difference CD spectrum of IIAGlc in the presence and absence of PG is shown in c. Pep18 is a synthetic peptide corresponding to the N-terminal 18 residues of IIAGlc (see Fig. 1). In a, green and red lines represent the CD spectra of Pep18 in phosphate buffer alone and in the presence of the anionic lipid PG, respectively. The black line is the CD spectrum of Pep18 in a mixture of the anionic E. coli lipids PG (25%) and PE (75%) (w/w) (total lipid concentration, 2 mM). In b, the black and red lines are the spectra of IIAGlc in the absence and presence of PG, respectively.

The interaction of intact IIA^Glc with PG was also investigated. The CD spectrum of IIA^Glc in phosphate buffer has a negative band at ~218 nm with shoulders at ~210 and ~224 nm (Fig. 2b, black), consistent with the known secondary structure of IIA^Glc, which comprises predominantly -strands with a few short helices (4-8). In the presence of PG, the CD spectrum of IIA^Glc became more negative with double minima at ~208 and ~222 nm (Fig. 2b, red), indicating -helix formation. The difference in the CD spectra of IIA^Glc in the presence and absence of PG (Fig. 2c) resembles the CD spectrum of Pep18 in the presence of PG (Fig. 2a, red) and can therefore be attributed to the conformational change of only the N-terminal segment of the protein. This interpretation is further supported by the observation that the migration of IIA^Glc in a nondenaturing gel is only slightly different in the presence or absence of PG, indicating that the overall shape of IIA^Glc remains essentially unaltered, and no global conformational change has taken place. Moreover, because the CD spectrum of IIA^Glc in the presence of PG is given by approximately the sum of the spectra of IIA^Glc without PG and Pep18 with PG, the amphipathic helical domain and the folded domain of IIA^Glc are independent of each other. Collectively, these data strongly suggest that the N-terminal segment of IIA^Glc is capable of binding to a negatively charged E. coli membrane surface.

The binding of the N-terminal segment of IIA^Glc to anionic phospholipids present in the membrane of E. coli suggests a two-state model for the structure of IIA^Glc (Fig. 3). In the cytosol, IIA^Glc is composed of the previously described globular core (residues 19-168, blue rectangle) and a disordered N terminus (residues 1-18, green tail) (4-8). In the second state, the N-terminal tail of IIA^Glc (residues 2-10) assumes a helical conformation (red) upon binding to the E. coli membrane via hydrophobic and electrostatic interactions, whereas the folded core domain interacts with the peripheral IIB domain of the membrane protein IIBC^Glc. Residues Asp¹³-Lys¹⁴-Lys¹⁵-Asp¹⁶-Thr¹⁷-Gly¹⁸ act as a linker between the two domains in the membrane-associated form of IIA^Glc. The proposed model provides a biological role for the amphipathic helical domain of cytoplasmic IIA^Glc. We suggest that effective phosphoryl transfer from IIA^Glc to IIBC^Glc requires the formation of a stable complex between the two proteins; the combination of the interaction of the folded domain (residues 19-168) with the IIB domain of IIBC^Glc and the helical domain (residues 2-10) with the membrane achieve this stability.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A two-state model for the functional role of the N-terminal domain of E. coli IIAGlc. The folded core domain of IIAGlc (residues 19-168) is represented by the blue box. The disordered and helical states of the N-terminal segment (residues 1-18) of IIAGlc are colored in green and red, respectively. Phosphoryl transfer involves formation of protein-protein complexes via contact between active sites (8, 26) as depicted here for IIAGlc-IIBCGlc. Attachment of IIAGlc to the E. coli membrane surface stabilizes the complex and consequently enhances its ability to donate the phosphoryl group to the IIB domain (circle), which is also on the membrane surface and covalently linked to the transmembrane domain IIC (rectangle).

It was previously shown that IIA^Glc could be cleaved at Lys⁷ by a membrane protease (21), consistent with the membrane association of the N-terminal domain of IIA^Glc. The clipped IIA^Glc (referred to as the "fast" form on account of its behavior during gel electrophoresis) has the same structured domain and ability to accept a phosphoryl group from HPr as does full-length IIA^Glc (known as the "slow" form) (6, 10, 21). However, the fast form of IIA^Glc is only 2-3% as active as the slow form in donating a phosphoryl group to the membrane protein IIBC^Glc (10). In light of the present data, we suggest that removal of the first seven N-terminal residues of IIA^Glc disrupts the amphipathic helix, making it a very poor membrane anchor.

Interestingly, IIA^Glc of Mycoplasma capricolum is cytoplasmic and contains a similar short N-terminal sequence, Met-Trp-Phe-Phe-Asn-Lys-Asn, which is rich in aromatic residues (italicized). Because aromatic residues can also play an important role in lipid binding (22), we propose that this segment of M. capricolum IIA^Glc has a similar membrane-anchoring role. Indeed, the N-terminal segment of M. capricolum IIA^Glc adopts an L-shaped structure (with the Trp sidechain disordered) in the crystal structure (23), which may be relevant for membrane binding as depicted in Fig. 3 for E. coli IIA^Glc. The difference in amino acid sequence, composition, and structure between the N-terminal membrane anchors of E. coli and M. capricolum IIA^Glc may reflect the lipid composition of the respective membranes. For example, cholesterol is one of the major lipid components of the membrane of M. capricolum (24).

Because the C terminus of IIA^Glc is adjacent to the N terminus with both ends being some 30 Å away from the active site (see Fig. 3 and Refs. 4, 5, and 8), the two positively charged residues at the C terminus (Lys¹⁶⁷ and Lys¹⁶⁸) of IIA^Glc may also participate in electrostatic interactions with the negatively charged E. coli membrane, further stabilizing the membrane-bound state (Fig. 3). Indeed, C-terminal truncation or mutation of the C-terminal basic residue to an acidic residue in the -glucoside permease from E. coli results in a 10-fold decrease in the catalytic rate of phosphoryl transfer (25).

The domain structure of the E. coli glucose transport system is IIA^Glc + IIBC^Glc, whereas that of some other sugar transport systems, for example that of mannitol, is IIABC, where all the domains are covalently linked and membrane-bound (1). Hence, those covalently linked IIAs are efficient phosphocarriers but are not available for other functions. The two states of IIA^Glc allow it to play multiple roles; it is a phosphocarrier in the PTS, as well as a regulator of a variety of other metabolic systems. The anchor function for the N-terminal helical domain defined here permits IIA^Glc to overcome the inherent defect in phosphotransfer capacity associated with its free-floating presence in the cytoplasm.

	ACKNOWLEDGEMENTS

We thank Drs. R. B. Cornell and R. J. Cushley (Simon Fraser University) for discussion and Drs. G. Christoph, D. Garrett, J. Louis, and B. Lee (National Institutes of Health) for assistance.

	FOOTNOTES

^* This work was supported in part by the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Chemical Physics, Bldg. 5, Rm. B1-30I, NIDDK, National Institutes of Health, Bethesda, MD 20892-0510. Tel.: 301-496-0782; Fax: 301-496-0825; E-mail: clore@speck.niddk.nih.gov.

Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.C000709200

² Unpublished data.

	ABBREVIATIONS

The abbreviations used are: IIX^Glc, enzyme IIX^Glucose; PTS, phosphoenolpyruvate:sugar phosphotransferase system; PG, dioleoylphosphatidylglycerol; PE, phosphatidylethanolamine; dioleolyl-PC, dioleolylphosphatidylcholine; Pep18, 18-residue synthetic peptide comprising the N-terminal 18 residues of IIA^Glc.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/51/39811    most recent C000709200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, G. Articles by Clore, G. M. Search for Related Content PubMed PubMed Citation Articles by Wang, G. Articles by Clore, G. M.